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Figure S3.
Further analysis of CMB localization, related toFig. 3. (A) Immunofluorescence micrograph showing CMB localizing to centrosomes in a subset of cells in Drosophila S2 cells. Quantitation of CMB localization reveals centrosome localization in ∼20% of cells. Pearson’s correlation coefficient analysis of centrosomal signal shows significant overlap between SAS-4 and CMB compared with randomized controls (single channel rotated by 90° with centrosome positioned in the upper right quadrant of the square analyzed). Error bars are the mean ± SD. N = 50 cells. Student’s t test was used; ****P < 0.0001. (B) Specificity of the polyclonal antibody raised against CMB confirmed by the absence of immunofluorescence signal in Cmb mutant testes. (C) Related to Fig. 3 I. Immunofluorescence micrographs showing some (CP110, ANA1, ANA2) but not all (SPD-2, γ-tubulin) centrosomal proteins colocalizing with CMB on cytoplasmic foci. (D) Pearson’s correlation coefficient analysis of cytoplasmic protein colocalization with CMB assessed on images as in C. Error bars are the mean ± SD. N = 50 cells per condition. A Mann–Whitney test was used to test statistical significance compared with randomized controls (single channel rotated 90°); ***P < 0.001, **P < 0.01. (E) Immunofluorescence micrographs showing SAS-4 colocalizing with Combover in the cytoplasm of primary spermatocytes in the testes. Colocalization was quantified by Pearson’s correlation coefficient analysis. Error bars are the mean ± SD. N = 12 animals. A t test was used to assess statistical significance of colocalization compared with randomized controls (single channel rotated 90°); ****P < 0.0001. (F) CMB-GFP colocalizes with PCM1 when expressed in HeLa cells. Colocalization was quantified by Pearson’s correlation coefficient analysis. Error bars are the mean ± SD. N = 193 cells. A t test was used to assess statistical significance of colocalization compared with randomized controls (single channel rotated 90°); ****P < 0.0001. (G) CMB-GFP expression fails to restore PCNT cytoplasmic centriolar satellite signal following depletion of endogenous PCM1 in HeLa cells. N = 40 cells analyzed per condition. Mean ± SD are displayed. A Mann–Whitney test was used to assess statistical significance; ****P < 0.0001, NS, not significant. Scale bars, 5 µm (A, C, F, and G), 10 µm (B), 1 µm (E, A, C, and F, insets).

Further analysis of CMB localization, related to Fig. 3,. (A) Immunofluorescence micrograph showing CMB localizing to centrosomes in a subset of cells in Drosophila S2 cells. Quantitation of CMB localization reveals centrosome localization in ∼20% of cells. Pearson’s correlation coefficient analysis of centrosomal signal shows significant overlap between SAS-4 and CMB compared with randomized controls (single channel rotated by 90° with centrosome positioned in the upper right quadrant of the square analyzed). Error bars are the mean ± SD. N = 50 cells. Student’s t test was used; ****P < 0.0001. (B) Specificity of the polyclonal antibody raised against CMB confirmed by the absence of immunofluorescence signal in Cmb mutant testes. (C) Related to Fig. 3 I. Immunofluorescence micrographs showing some (CP110, ANA1, ANA2) but not all (SPD-2, γ-tubulin) centrosomal proteins colocalizing with CMB on cytoplasmic foci. (D) Pearson’s correlation coefficient analysis of cytoplasmic protein colocalization with CMB assessed on images as in C. Error bars are the mean ± SD. N = 50 cells per condition. A Mann–Whitney test was used to test statistical significance compared with randomized controls (single channel rotated 90°); ***P < 0.001, **P < 0.01. (E) Immunofluorescence micrographs showing SAS-4 colocalizing with Combover in the cytoplasm of primary spermatocytes in the testes. Colocalization was quantified by Pearson’s correlation coefficient analysis. Error bars are the mean ± SD. N = 12 animals. A t test was used to assess statistical significance of colocalization compared with randomized controls (single channel rotated 90°); ****P < 0.0001. (F) CMB-GFP colocalizes with PCM1 when expressed in HeLa cells. Colocalization was quantified by Pearson’s correlation coefficient analysis. Error bars are the mean ± SD. N = 193 cells. A t test was used to assess statistical significance of colocalization compared with randomized controls (single channel rotated 90°); ****P < 0.0001. (G) CMB-GFP expression fails to restore PCNT cytoplasmic centriolar satellite signal following depletion of endogenous PCM1 in HeLa cells. N = 40 cells analyzed per condition. Mean ± SD are displayed. A Mann–Whitney test was used to assess statistical significance; ****P < 0.0001, NS, not significant. Scale bars, 5 µm (A, C, F, and G), 10 µm (B), 1 µm (E, A, C, and F, insets).

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